SUMMARY

The sticky spiral of araneoid spider orb webs consists of silk fibers
coated with adhesive droplets. The droplets contain a variety of
low-molecular-mass compounds (LMM). Within a species, a fairly consistent
ratio of LMM is often observed, but substantial variability can exist. To gain
insight into factors influencing LMM composition, spiders of three araneid
species were starved and LMM from their webs were analyzed for changes in
composition. To determine if these changes were consistent with the spider's
ability to synthesize the different organic LMM, synthetic capacities were
estimated following the feeding of radiolabeled metabolites. Some changes in
droplet composition were broadly consistent with differing synthetic
capacities: molar percentages of less readily synthesized compounds (e.g.
choline, isethionate, N-acetyltaurine) typically declined with
starvation, at least during a portion of the imposed fast, while more readily
synthesized compounds (e.g. GABamide, glycine) tended to increase. Most
striking was the apparent partial substitution of N-acetylputrescine
by the more readily synthesized GABamide in fasting Argiope
trifasciata. However, departures from expected compositional shifts
demonstrated that synthetic capacity alone does not adequately predict sticky
droplet compositional shifts with starvation. Moreover, feeding controls
exhibited some changes in composition similar to starving spiders. As the webs
of both feeding and starving spiders were removed for chemical analysis and
could not be recycled, the loss of LMM contained in these webs likely
contributed to similarities between treatments. In addition, feeding spiders
molted, oviposited and/or built heavier webs. The added metabolic demands of
these activities may have contributed to changes in composition similar to
those resulting from starvation.

The LMM composition of sticky droplets often differs quantitatively and
qualitatively among araneoid species
(Vollrath et al., 1990;
Townley et al., 1991;
Higgins et al., 2001) (M. A.
Townley and E. K. Tillinghast, unpublished). The significance of these
differences is unknown. One attempt to determine if differences in composition
translate into differences in web hygroscopicity among three araneid species
did not demonstrate such a relationship
(Townley et al., 1991). Nor do
we know the extent to which the composition of the droplets is tailored to the
physical environment in which a spider forages, the prey captured, or the
metabolic needs of the spider. And while LMM composition within a species is
consistent enough that analyses of pooled web collections from groups of
individuals generally yield similar results
(Vollrath et al., 1990;
Townley et al., 1991),
substantial intraspecific differences have also been observed within and among
populations, between the sexes, and following a change in environment/diet
(Vollrath et al., 1990;
Townley et al., 1991;
Higgins et al., 2001).

The goal of the present study was to examine the influence of starvation on
the LMM composition of the sticky droplets and to determine if observed
changes reflect differences in the spider's capacity to synthesize the various
organic LMM. Specifically, we anticipated that the total mass of LMM would
decrease in webs of starving spiders, but that there would be greater relative
declines in those organic LMM the spider is less able to synthesize.
Therefore, in addition to analyzing series of webs built by starving and
feeding spiders of three araneid species, we fed radiolabeled compounds to two
of these species to determine to what extent the spiders can synthesize the
different organic LMM (Kasting and
McGinnis, 1966). Given the results of these synthetic capacity
measurements, some of the changes in composition observed with starvation
conformed to our expectations, but others did not. Unexpected results also
came from the control feeding spiders, which also exhibited changes in droplet
composition, in some respects similar to trends seen in webs of starving
spiders. Possible explanations for these results are discussed. In addition,
the construction of egg sacs by some of the study spiders allowed us to make a
preliminary examination of the influence of the egg laying cycle on droplet
composition.

Materials and methods

Synthesis of organic LMM by Argiope

Spider collection, maintenance and radioisotope feeding

Adult female Argiope aurantia Lucas 1833 and Argiope
trifasciata (Forskål 1775) were collected in southern New
Hampshire, USA from late July to late August and in the latter half of
September, respectively. Spiders were housed individually in wood or aluminum
frames (51 cm×51 cm×9 cm) with glass plates on the front and back.
They were exposed to the prevailing natural light:dark cycle in a room facing
east at temperatures and relative humidities that approximated outside
conditions.

Four groups of A. aurantia (5–7 spiders/group) and three
groups of A. trifasciata (6–7 spiders/group) were formed. For
each A. aurantia, 1.1×105 Bq
d-[U-14C]glucose (ICN Biomedicals, Irvine, CA, USA) in 3μ
l water was placed on the mouthparts using a 10 μl Hamilton syringe
(Reno, NV, USA) and the spider was observed until the droplet was completely
imbibed. Argiope trifasciata were likewise fed
d-[U-14C]glucose, but in quantities of
3.7×104–1.1×105 Bq/spider.

Spiders were either fed radioisotope within 3 days of being captured or, if
held longer prior to isotope feeding, were fed crickets or grasshoppers. They
were not fed after isotope feeding, but orbs built on the day of isotope
feeding were partially collapsed and the spiders were allowed to recycle them.
Water was given daily, except for the day before and the day of isotope
feeding to encourage drinking of the labeled solution.

Handling of radiolabeled orb webs

The first five webs built by each spider after ingesting radioisotope were
collapsed, wound onto one end of a glass micropipet, and stored at–
20°C. All webs built by members of the same group were pooled on a
single micropipet (19–30 webs/group). Not all spiders built five webs.
While still on their micropipets, each group's web collection was extracted
twice in 4 ml distilled water for 1.5 h with occasional gentle vortexing. The
two extracts were pooled, dried and analyzed by proton nuclear magnetic
resonance spectroscopy (1H-NMR) as described below using the
Argiope acquisition parameters (see `1H-NMR analysis').
From these analyses molar percentages of the 11 organic LMM (listed in the
Introduction) in each of the 11 extracts (seven 14C-labeled, four
35S-labeled) were calculated. Extracts were then fractionated
individually by high-voltage paper electrophoresis (HVE).

HVE and chromatography of radiolabeled water-soluble web
fractions

Each water-soluble extract was fractionated by HVE at 3000 V on 23
cm×57 cm sheets of Whatman 3MM chromatography paper (Brentford,
England), with the extract applied in 75 μl of electrolyte solution over a
13 cm long origin. For the 14C-labeled extracts, the origin was 28
cm from the positive pole and electrophoresis was carried out for 35 min. For
the 35S-labeled extracts, the origin was 41 cm from the positive
pole and the run lasted 55 min. The electrolyte solution used was
pyridine:glacial acetic acid:water (133:4.6:1862.4, v/v), pH 6.4. Coolant at
2°C was passed through the unit's lower plate.

After electrophoresis, the paper was air dried and autoradiograms were
generated using Kodak BioMax MR film (Rochester, NY, USA). Using the
autoradiograms as a guide, the electrophoretograms were cut into radioactive
and nonradioactive bands. All bands were eluted overnight with distilled water
containing 0.001% sodium azide in a chamber saturated with water vapor.
Eluates were dried, resuspended in 1 ml distilled water, and 50 μl removed
for scintillation counting. Selected eluates were examined by
1H-NMR to establish locations of organic LMM.

To determine what percentage of the radioisotope in a HVE eluate was
incorporated into an organic LMM of interest, portions of radioactive eluates
were further fractionated by two-dimensional thin layer chromatography
(2D-TLC) on 20 cm×20 cm cellulose plates (0.1 mm thickness, Merck KGaA,
Darmstadt, Germany) using pyridine:acetone:ammonium hydroxide:water
(45:30:5:20, v/v) in the first dimension and 2-propanol:formic acid:water
(75:12.5:12.5, v/v) in the second dimension
(Schmidt, 1974). This was
necessary because HVE did not resolve all of the organic LMM measured in this
study and because these compounds, though they constitute the bulk of the
organic LMM, could not be assumed to be the only organic or sulfur-containing
compounds extracted by water from orb webs (and, indeed, they are not).
Autoradiograms were prepared from the 2D-TLC plates as above. If an
autoradiogram revealed two or more radioactive compounds, these were
individually scraped off the plate and their radioactivities measured by
scintillation counting. Identifications of the organic LMM on the plates were
based on NMR of the HVE eluates and on experience in this system with the
migration characteristics of most of the organic LMM of interest. Where any
doubt remained, radioactive compounds were scraped off plates and examined by
NMR to confirm identity.

Using pre- and post-fractionation NMR data and the radioactivity data
obtained following HVE and 2D-TLC, relative specific radioactivities for the
organic LMM were estimated (initially in c.p.m./mole%, then, for
14C-labeled LMM, converted to c.p.m./molar quantity of carbon).

For the two N-acetylated LMM, NAP and NAT, estimates were also
made of the relative specific activities of their acetyl groups
versus their Put or Tau moieties. Because NAP was not detected in
webs of A. aurantia, only the three A. trifasciata
water-soluble extracts were used to make this determination for this compound.
After identifying the HVE eluates containing NAP and NAT by NMR, 70% of each
was hydrolyzed under vacuum (6.7 Pa) in 6 mol l–1 HCl at
115°C for 20 h. At the same time, commercial samples of Put and Tau (two
each) were treated likewise. Based on the percentage recovery of these
standards (Put, 83.8%; Tau, 84.3%), a correction for losses occurring during
hydrolysis was made. Hydrolyzed and unhydrolyzed portions of eluates were
fractionated by HVE and 2D-TLC and autoradiograms generated. Of the total
14C present in these eluates, the percentage incorporated into the
Put/Tau moieties versus the intact compounds was determined by
scintillation counting of compounds scraped off the 2D-TLC plates. The
difference between these was taken to be the percentage of the 14C
incorporated into the acetyl groups.

Effects of starvation and web removal on orb web mass and LMM
composition in Araneus and Argiope

Collection and maintenance of spiders

Juvenile Araneus cavaticus (Keyserling 1882) were collected from
barns in southern New Hampshire, USA between early April and mid-July.
Juvenile female Argiope aurantia and Argiope trifasciata
were also collected locally from mid-July to early August and throughout
August, respectively. Spiders were housed as described above.

Before spiders molted and were placed in an experimental group, they were
fed 1–3 flies on days they built webs, were allowed to recycle (i.e.
consume) their webs freely, and were given water daily. Both before and after
assigning spiders to groups, A. cavaticus and A. aurantia
were fed house flies (Musca domestica L.) and other dipterans
[primarily Phaenicia sericata (Meigen 1826) and Phormia
regina (Meigen 1826)], while A. trifasciata received house flies
exclusively.

Formation of study groups and collection of orb webs

During this study, 21 groups were formed (supplementary material Fig. S1),
with the spiders composing a group receiving the same treatment (fed or
starved) and being of the same species, sex and stage (juvenile or adult) and,
for adult females, having made the same number of egg sacs (0–3). All
spiders were added to a group at the same point in the molt/intermolt cycle
(beginning of intermolt). With A. cavaticus, webs of male and female
juveniles (all penultimate instars) and adult females were collected. With the
two Argiope species, only webs built by adult females were collected.
An individual spider could belong to only one group at a time, but to two or
more groups over the course of the study, as explained below.

Orb webs were collected from a spider only once it had reached the desired
stage following its most recent ecdysis. From 1–3 molts were required in
the laboratory before the desired stage was reached. Spiders were initially
divided into a feeding group and a starving group, with the first individual
to reach the desired stage randomly assigned to one of these groups. The next
individual that molted to the desired stage was then assigned to the other
group and this alternation continued as other spiders subsequently molted to
the desired stage.

Following ecdysis, with the building of the first orb web, the spider,
whether assigned to a feeding or starving group, was fed two or more flies
(see above) totaling about 50 mg wet mass. The web was then partially
collapsed and the spider was allowed to recycle it. Thus, no compositional or
mass data were obtained from the first web. All subsequent webs, however, were
collected on micropipets and stored at –20°C. All second webs built
by members of the same group were collected on the same micropipet and are
referred to as the web 2 collection of the group. Likewise, all third webs
built by the spiders within a group were pooled to yield the web 3 collection
and so on. The complete set of web collections from a group is referred to as
a `series'.

Spiders in starving groups were treated differently from spiders in feeding
groups after the construction of the second orb web. In both groups, the
second webs were collected as described above, but then only feeding group
spiders were handed one or more flies, totaling about 25–35 mg wet mass.
Feeding group spiders were fed only on days they built a web. All spiders were
given water daily. With starving groups, spiders were stressed until resultant
changes in behavior/physiology were readily apparent (e.g. sluggishness,
construction of incomplete webs), then feeding was resumed, at which point the
spiders became members of a `resumed feeding' group (supplementary material
Fig. S1).

All constructed orb webs, unless they contained no sticky spiral
whatsoever, were collected from each member of a group and added to that
group's series of web collections until one of four events occurred
(supplementary material Fig. S1): (1) the spider was transferred to another
group, (2) the spider died, (3) the spider escaped, or (4) the date arrived,
30 September, on which other obligations forced us to end web collecting for
the year. This lattermost event was not too detrimental as many of the spiders
involved, all adult A. cavaticus or A. aurantia, had already
become relatively inactive by this time and would have built few additional
webs. However, the series from the A. aurantia PES3 group (see next
paragraph) was ended prematurely. Argiope trifasciata webs were
collected during a different year when there was no need to terminate the
study on a particular date.

There were three reasons for transferring a spider to another group.
Transfer of starving group spiders to a resumed feeding group has already been
described. The other two reasons were egg sac construction and molting. Egg
sac construction resulted in a spider being transferred to a post-egg-sac
(PES) group. Because some individuals of both A. aurantia and A.
trifasciata produced three egg sacs (containing nonviable eggs), the
construction of each of which was followed by the spinning of additional orb
webs, there are three successive PES groups for both species (PES1, 2 and 3;
supplementary material Fig. S1). Molting pertains to the penultimate instar
A. cavaticus only since adults do not molt. On reaching adulthood
males lack the ability to build orb webs
(Sekiguchi, 1955) so the final
molt marked the end of their period of service. Female A. cavaticus,
on the other hand, by the procedure described above, were assigned to either a
feeding adult or a starving adult group after their final molt (supplementary
material Fig. S1).

Following the transfer of a spider to either a resumed feeding or PES group
we did not allow it to recycle its first web, as spiders in starving or
feeding groups were allowed, but collected this web and fed the spider one or
more flies totaling about 25–35 mg wet mass. In all other ways web
collection and feeding were carried out as for members of feeding groups.

Gravimetric measurements of orb webs

Each web collection was scraped off its micropipet with a razor blade,
desiccated in vacuo over phosphorus pentoxide for at least 2 days,
weighed to the nearest 0.01 mg, then extracted three times in 2 ml distilled
water for 1 h with occasional gentle vortexing. The three extracts were pooled
and dried on a Savant Speed Vac Concentrator (Hicksville, NY, USA), then
transferred with two volumes of distilled water totaling 300 μl to a
pre-weighed cup fashioned from the cap of a microcentrifuge tube. After
desiccating as above for at least 3 days, this water-soluble fraction of the
web, containing the LMM, was weighed and then transferred back to its sample
tube with three volumes of distilled water totaling 1 ml. After drying, the
sample was analyzed by 1H-NMR. The desiccated water-insoluble
fraction was also weighed.

Within a group, not all individuals built the same number of webs
(supplementary material Table S1). Consequently, later web collections in a
series contained fewer webs than earlier web collections. Initially,
therefore, anticipating difficulty with 1H-NMR analysis on the
smaller samples (due to unacceptably large numbers of scans being required to
achieve a good signal-to-noise ratio), we typically pooled two or more
end-of-series web collections to generate water-soluble extracts that would be
large enough to allow for analysis in a reasonable amount of time. Note that
this more infrequent `horizontal pooling' of, e.g. web collections
16–19, is in addition to the invariable practice of `vertical pooling'
of, e.g. all second webs built by the members of a group. We found, however,
that our first horizontal poolings were too generous and that smaller samples
(0.3–1 mg) could be analyzed within an acceptable amount of time (3 h to
overnight). Thus, we did less horizontal pooling as the study progressed. As
the first webs analyzed were those built by Araneus cavaticus, more
horizontal pooling was carried out on webs of this species.

Gravimetric measurements of spiders

Fresh and dry mass data on the three study species were obtained to gain
some measure of the percentage of a spider's dry mass that it typically
invests in its orb web, thus providing an indication of the loss incurred
when, as in this study, a spider is denied the opportunity to recycle its web.
Freshly captured local penultimate instar and adult female Araneus
cavaticus, and adult female Argiope aurantia and Argiope
trifasciata were weighed immediately on return to the laboratory. Some of
these individuals were anesthetized with CO2, immersed in 80%
ethanol for 1 h, and dried to constant mass in a 43°C oven. Only adult
females that were not conspicuously gravid were collected for these
measurements.

Composition of orb web water-soluble fractions

1H-NMR analysis. The water-soluble fraction of each web
collection was dissolved in 0.5–1.0 ml 99.96% D2O (Cambridge
Isotope Laboratories, Andover, MA, USA) and analyzed on a Bruker AM-360
spectrometer (Billerica, MA, USA) with a 5 mm proton selective probe. 360-MHz
1H spectra were obtained at a temperature of 300K with
2-methyl-2-propanol added as an internal standard (δ=1.2200 p.p.m.).
Following Fourier transformation, peak areas were integrated and used to
calculate the molar percentages of the 11 organic LMM dealt with in this
study. 1H-NMR chemical shifts and coupling constants for each of
these LMM in D2O have been reported previously
(Townley et al., 1991;
Higgins et al., 2001).

In all NMR analyses a spectral width of 5000 Hz was examined and a pulse
width of 4.3 μs, yielding about a 53° flip angle, was used. Various
numbers of scans (128–5064) were accumulated depending on sample size.
Several other acquisition parameters differed between A. cavaticus
and Argiope samples. With the A. cavaticus extracts,
analyzed first, the pulse repetition time was the same as the acquisition
time, 3.28 s, during which 32K data points were acquired and later zero-filled
to 64K prior to Fourier transformation. However, analyses of a standard
solution containing the 11 organic LMM indicated that a longer time between
pulses would yield more accurate molar percentages (supplementary material
Table S2), particularly for Pro. Therefore, with the Argiope
extracts, a longer pulse repetition time was adopted (8.28 s) that still
allowed samples to be analyzed within a reasonable amount of instrument time,
with 64K data points acquired over 6.55 s, later zero-filled to 128K. We did
not attempt to apply corrections to the A. cavaticus data. Thus, more
than anything else, Pro is likely somewhat underrepresented in the A.
cavaticus webs, though consistently so.

In addition to measuring relative quantities of the organic LMM in molar
percentage, we wanted to estimate absolute quantities of the organic LMM. With
the A. cavaticus web collections, this was achieved by a standard
addition method whereby each sample was analyzed by NMR twice, before and
after the addition of synthetic GAB
(Kleemann et al., 1980) (1μ
mol mg–1 water-soluble fraction). Integrations from the
two analyses allowed us to calculate average μg web–1 for
each organic LMM in each web collection. We also calculated the percentage of
the water-soluble fraction's mass that could be accounted for by the 11
organic LMM. For the two Argiope species, we obtained approximateμ
g web–1 of the organic LMM by assuming that the
percentage of the water-soluble fraction's mass that could be accounted for by
the 11 organic LMM was the same as the mean in A. cavaticus.

Phosphorus analysis. After NMR analysis, each water-soluble web
fraction was assayed for inorganic phosphorus
(Chen et al., 1956) using
KH2PO4 as a standard.

Statistical analyses

Pearson correlation coefficients were calculated from molar percentage
data: (1) for all pairwise combinations of individual LMM, with data from all
non-radioactive web collections from each species pooled for the analyses, (2)
between H2PO –4 and total positively
charged LMM, total negatively charged organic LMM, and `excess' positive
charge (see `H2PO –4 and charge
balance' in Results), and (3) for all pairwise combinations of web collection
number and total readily synthesized, moderately synthesized, and poorly
synthesized LMM (see `Synthesis of organic LMM by Argiope' in
Results) in web collections from the starving and feeding groups. As in an
earlier study (Higgins et al.,
2001), all molar percentages were arcsine-transformed prior to
analysis and Bonferroni-corrected P values are reported.

Linear regression analysis was used to evaluate changes in three quantities
over the series of web collections for a group: (1) the percentage of web mass
that was solubilized by water, (2) the percentage of the water-soluble
fraction's mass that was accounted for by the 11 organic LMM in A.
cavaticus (see `1H-NMR analysis' above), and (3) the `excess'
positive charge. A t-test was used to determine if slopes of
regressions differed significantly from zero. Slopes of `excess' positive
charge regressions were compared using a Tukey's multiple comparison test
(Zar, 1999).

Specific radioactivities of LMM from radiolabeled webs were normalized as
described in Table 1,
log10-transformed, and compared using analysis of variance (ANOVA)
and Tukey's HSD multiple comparisons test. The specific radioactivities
reported in Table 1 are
back-transformed means and 95% confidence intervals.

Molar percentages and relative specific radioactivities of organic LMM
in water extracts of radiolabeled orb webs from Argiope fed
[U-14C]glucose or a solution containing
L-[35S]methionine and L-[35S]cysteine

Results

Synthesis of organic LMM by Argiope

Relative specific radioactivities of the organic LMM from the labeled web
collections are presented in Table
1, along with the molar percentages of the organic LMM in these
webs. Put and free Pro were not detected by NMR in any of the radiolabeled web
collections. Thus, specific activity data were not obtained for these
compounds, though the specific activity of the Put component of NAP was
measured. The specific activity of free Ala was determined only from the
A. trifasciata web extracts because of the very small amounts of Ala
in the A. aurantia extracts. Specific activities for Bet and
14C-labeled Tau were not obtained because we were not convinced
these compounds had been adequately resolved. However, relative specific
activities were determined for 35S-labeled Tau and for the Tau
moiety of 14C-labeled NAT.

In 14C-labeled webs, the specific activity of NAT was
significantly higher than that of Ise
(Table 1). Hydrolysis of NAT,
however, revealed that the bulk of the 14C incorporated into this
compound was restricted to its acetyl group and that the specific activities
of Ise and the Tau moiety of NAT were not significantly different. Neither
were the specific activities of NAT and Ise significantly different in
35S-labeled webs. In contrast to NAT, the label in NAP was more
evenly divided between the acetyl and Put moieties, though on a per mole
carbon basis, the acetyl group's specific activity was again higher than that
of Put, though not significantly so. This difference between the two
N-acetylated compounds was in greater measure due to the
significantly higher specific activity of the acetyl group in NAT as compared
with the acetyl group in NAP, and to a lesser extent due to the higher (though
not significantly so) specific activity of the Put moiety as compared with the
Tau moiety. Recall that comparisons between these two LMM could be made only
in A. trifasciata since NAP was not detected in A.
aurantia.

Based on the specific radioactivities obtained, we divided the LMM in
Table 1 into three categories:
readily synthesized LMM (Ala, Gly, GAB), moderately synthesized LMM (NAP, Put,
NAT, Tau, Ise), and poorly synthesized LMM (Cho, Bet). Assuming the acetyl
moieties of NAP and NAT can be readily synthesized, LMM with mean specific
activities greater than that of the acetyl group of NAP were taken to be
readily synthesized. As we did not obtain specific activities for Pro, its
placement is uncertain, but it is likely at least a moderately synthesized
compound. Free Put and Tau were assumed to have specific activities comparable
to those of the Put and Tau moieties of NAP and NAT, respectively, and it was
the data for these moieties that dictated the inclusion of NAP and NAT in the
moderately synthesized category. Though we also did not obtain data for Bet,
we tentatively consider it a poorly synthesized LMM, given earlier
observations (Higgins and Rankin,
1999) and the likelihood of its synthesis from Cho (see
Discussion).

We predicted that, with starvation, molar percentages of readily
synthesized LMM would tend to increase while those of poorly synthesized LMM
would decrease and these trends would be reversed when feeding was resumed.
The fate of moderately synthesized LMM was more uncertain, but our suspicion
was that their molar percentages would either decrease or just be maintained
with starvation.

Molar percentages of organic LMM were measured in the radiolabeled web
collections simply to allow us to express radioisotope incorporation in terms
of relative specific radioactivities. We assumed LMM compositions in these
webs would be similar to those in some of the non-radioactive web collections.
In some respects they were, but there were also differences. The molar
percentages of Cho and Bet are especially noteworthy in this regard. The
lowest molar percentage of Cho in the eight radiolabeled A. aurantia
web extracts (10.9 mole%) was higher than the highest molar percentage of Cho
in the 102 unlabeled A. aurantia web extracts (8.9 mole%). (Note that
these two molar percentages were calculated based on the 11 organic LMM
constituting 100% of the LMM. The highest molar percentage for Cho in A.
aurantia given in Table 2
is 7.4 mole% because the percentages in that table were calculated based on
the 11 organic LMM plus H2PO4–
constituting 100% of the LMM.) Likewise, the lowest molar percentage of Cho in
the three labeled A. trifasciata web extracts (22.6 mole%) was higher
than the highest molar percentage of Cho in the 123 unlabeled A.
trifasciata web extracts (13.9 mole%). The same sort of discrepancy,
albeit not as extreme, was also observed in A. aurantia with regard
to molar percentage of Bet. Unlabeled webs were built by adult female
Argiope maintained on a diet of flies. They were collected as
juveniles and raised in the laboratory through 1–2 molts and were
unmated. Labeled webs were built by adult female Argiope that had
recently been feeding in the field or, if fed in captivity, were given
crickets or grasshoppers. They were collected as adults and were likely
mated.

Relative quantities (in mole%) of the organic LMM and inorganic
phosphate in orb webs of the three species studied, as measured in
non-radioactive web collections

Effects of starvation and web removal on orb web mass and LMM
composition

The number of webs composing each web collection is given in supplementary
material Table S1.

Gravimetric data

Mass per orb web

Withholding prey and removing webs resulted in an immediate drop in mean
orb web mass in four of the five starving groups and the downward trend
continued until feeding was resumed (Fig.
1, supplementary material Figs S3–S5). Only in the
Argiope aurantia starving adult group was there a slight delay before
web mass likewise declined (supplementary material Fig. S6). When feeding was
resumed, web mass quickly increased, though, among adults, Argiope
trifasciata were slower to recover web mass than A. aurantia or
Araneus cavaticus. Among A. cavaticus, juvenile males and
females were slower to recover web mass than adult females. Web mass among
resumed feeding adults, but not juveniles, ultimately returned to or exceeded
the mass at the start of the stadium.

Among feeding group juvenile A. cavaticus, web mass was maintained
or slightly increased in the earlier part of the stadium for both females and
males. As they approached their final molt, web mass for females remained high
(supplementary material Fig. S4), while those for males declined
(supplementary material Fig. S3). Among feeding adult and A. aurantia
PES groups, an upward trend followed by a decline in the days leading up to
egg sac construction was typical (Fig.
1, supplementary material Figs S5–S7). In the A.
trifasciata PES groups, an initial upward trend was less in evidence, but
decreases in web mass prior to construction of the next egg sac were observed
(supplementary material Fig. S7). Web mass in the three resumed feeding adult
groups, however, did not tend to drop near the end of the series
(Fig. 1, supplementary material
Figs S5, S6).

Web mass as a percentage of spider mass

Table 3 presents data on
fresh masses of the three species of spiders and their dry masses fractions.
It also presents desiccated masses of webs built by feeding group spiders and
the total masses of the 12 measured LMM in these webs. From these data we
estimate that these spiders typically invested about 1–3% of their dry
mass into the materials composing an orb web and about 0.5–1% of their
dry mass into the 12 measured LMM per web.

Water-soluble percentages

For A. cavaticus, the percentage of the desiccated web mass
extracted by water (mean ± s.d.) was 71.0±4.24%, N=86
web collections (juveniles 72.4±4.06%, N=51; adults
68.9±3.57%, N=35). For A. aurantia and A.
trifasciata, as observed previously
(Townley et al., 1991),
water-soluble percentages were usually lower, means ± s.d.
54.6±5.70% (N=105) and 52.8±7.45% (N=123),
respectively. Water-soluble fractions undoubtedly include some non-LMM
components, such as nodular glycoprotein (see Introduction). Very low
water-soluble percentages, far below the averages, were seen in some web
collections built at or near the end of the series in the A.
trifasciata feeding, resumed feeding, PES2 and PES3 groups, and in the
A. aurantia PES1 group. It is likely that additional examples of very
low water-soluble percentages were masked as a result of end-of-series
`horizontal' pooling (see `Gravimetric measurements of orb webs' in Materials
and methods and supplementary material Table S1).

Water-soluble percentages tended to decrease over the series. Slopes of
linear regressions differed significantly from zero in 14 of the 21 groups,
and these 14 slopes were all negative (range: –0.22 to –1.65) and
spread across all species and treatments (supplementary material Table
S3).

The percentage of the water-soluble mass accounted for by the 11 organic
LMM in A. cavaticus was 57.8±5.4% (mean ± s.d.,
N=86). There was a tendency for this percentage to decrease over the
series irrespective of treatment, but linear regression slopes departed
significantly from zero in only three of the nine groups of A.
cavaticus (feeding juvenile females, P=0.005; starving adult
females, P=0.030; resumed feeding adult females, P=0.030).
When the data from all nine groups were pooled, the slope of the regression
was significantly different from zero (P=0.039), with an average
decrease from one web collection to the next of 0.27%.

LMM compositional data

Molar percentage data (averages and ranges) for the 11 organic LMM and
inorganic phosphate in the non-radioactive webs of each species are summarized
in Table 2. These data were
also analyzed for correlations between LMM
(Table 4). Some significant
correlations showed agreement among our three species and/or with Nephila
clavipes L. (Higgins et al.,
2001) and these are indicated in
Table 4.

Pearson correlation matrices for arcsine-transformed molar percentages
of the 12 LMM in all non-radioactive web collections from each
species

Changes in molar percentages of eight of the organic LMM with starvation
and subsequent resumed feeding are shown in Figs
2,
3,
4.
Fig. 2 contains the readily
synthesized LMM, Ala, Gly, and GAB, Fig.
3 the three most abundant moderately synthesized LMM, NAP, NAT and
Ise, and Fig. 4 the poorly
synthesized LMM, Cho and Bet (see `Synthesis of organic LMM by
Argiope' above). These figures also present the corresponding data
from the feeding groups.

Bar charts showing both the absolute and relative quantities of all 12
measured LMM in web collections from the A. trifasciata starving,
resumed feeding, and feeding groups are presented in
Fig. 1. supplementary material
Figs S3–S6 show the corresponding bar charts for A. cavaticus
and A. aurantia (key in supplementary material Fig. S2). Absolute and
relative quantity data for the 12 LMM in each web collection from all groups
are also available in tabular form in supplementary material Tables
S4–S6.

Absolute (A,B) and relative (C,D) quantities of LMM in water extracts of
webs built by Argiope trifasciata adult females in feeding (A,C) and
starving/resumed feeding (B,D) groups. Average desiccated web masses within
web collections are indicated by line graphs in A and B (right
y-axis). The key (Bii) identifies the LMM, arranged in the same
order, represented in all six bar charts. Several major LMM are also directly
labeled in Di to further aid orientation. LMM with a net charge are
represented by solid black or white, while net neutral LMM are shown in color.
Proline was not detected in any A. trifasciata web collections. GAB,
GABamide; Gly, glycine; Ise, isethionate; NAP,
N-acetylputrescine.

In all tables and figures except Table
1, molar percentages were calculated based on the 12 LMM
constituting 100% of the LMM. (In Table
1, molar percentage was calculated based on the 11 organic LMM
constituting 100% of the LMM.) In all three species, however, several
as-yet-unidentified and generally minor organic LMM were detected in many of
the spectra. There are undoubtedly other inorganic LMM components as well,
such as NO –3, K+, Na+ and
Ca2+ reported from webs of other araneoid species
(Schildknecht et al., 1972;
Work, 1981;
Tillinghast and Christenson,
1984; Patel and Nigam,
1996).

Organic LMM

We predicted that molar percentages of Ala, Gly and GAB would increase with
starvation and then drop when feeding was resumed. Conforming to expectations,
percentages of Gly and GAB increased with starvation in all three species, at
least in the earlier part of the series
(Fig. 2). It is especially
noteworthy that not only molar percentages, but also absolute quantities of
Gly and GAB increased in the webs of starving spiders; Gly in all three
species and GAB in Argiope trifasciata, again, at least in the first
portion of the series (Fig. 1,
supplementary material Figs S3–S6 and Tables S4–S6). With resumed
feeding, molar percentages of Gly immediately dropped in all three species, as
did GAB in A. trifasciata, though, unexpectedly, they again tended to
increase as the resumed feeding series progressed. Other trends were also not
expected: free Ala remained a minor component in starving Araneus
cavaticus and Argiope aurantia, increasing only slightly
(indeed, less than in feeding groups), and in A. trifasciata Ala
clearly declined with starvation (Fig.
2). Also, GAB did not decrease with resumed feeding in A.
cavaticus and A. aurantia. Even more unanticipated, increases in
Gly and GAB were also seen in the feeding groups, where more stable
compositions had been expected. These increases are reflected in positive
correlations in feeding groups between web collection number and the summed
molar percentages of the three readily synthesized LMM, very similar to
correlations seen in the starving groups
(Table 5).

Pearson correlation matrices describing relationships among web
collection number and arcsine-transformed summed molar percentages of readily
synthesized, moderately synthesized and poorly synthesized LMM in the starving
and feeding groups of each species

We suspected that the moderately synthesized LMM would decrease or at most
be maintained in starving spiders. The low specific activities of Ise and NAT
(Table 1) made it especially
likely that they would decline. With starvation we observed trends toward
decreased molar percentages of not only Ise and NAT, but NAP as well
(Fig. 3). In starving A.
cavaticus females, however, decreases in NAT were preceded by increases
such that percentages of NAT at the end of the series were similar to those at
the beginning. Starving spiders responded to resumed feeding with increased
molar percentages of NAP, NAT and Ise, though subsequent trends varied as the
resumed feeding series progressed and include examples of declines in molar
percentages after the initial rise (e.g. Ise in all three species) as well as
more prolonged upward trends (e.g. NAT in A. trifasciata and A.
cavaticus). Interestingly, in contrast to the rapid and substantial
recovery of Ise in resumed feeding A. trifasciata, resulting in high
molar percentages of Ise relative to feeding group A. trifasciata,
NAT's recovery was slow and modest, resulting in low percentages of NAT
relative to the feeding group (Figs
1,
3).

In agreement with expectations, molar percentages of NAP were essentially
maintained by feeding spiders and, while not necessarily maintained at a
constant level, percentages of NAT in webs of feeding spiders were generally
higher than in the corresponding webs of starving spiders. Ise, however,
exhibited molar percentage declines in webs of feeding spiders that were
sometimes very similar to those of starving spiders. These contributed to
negative correlations between web collection number and summed molar
percentages of the moderately synthesized LMM in feeding groups, though these
correlations were less significant than those of the starving groups
(Table 5). Negative
correlations between total readily synthesized and moderately synthesized LMM,
and positive correlations between moderately synthesized and poorly
synthesized LMM, in both starving and feeding groups, indicate that the
moderately synthesized LMM generally exhibit shifts in relative abundance more
like poorly synthesized, rather than readily synthesized, LMM
(Table 5). Several correlations
between NAT and other LMM (Table
4), however, demonstrate that this is not invariably the case
(though Table 4, unlike
Table 5, considers data from
resumed feeding and PES groups as well as from starving and feeding
groups).

Cho and Bet were expected to decline with starvation and, indeed, declines
were observed in all three species (Fig.
4). But as with GAB, Gly, and Ise, similar trends were also
unexpectedly exhibited by the feeding groups
(Table 5). Moreover, in the
starving groups, after a steep initial decline, Cho tended to level off (or
partially rebound) at molar percentages that exceeded the percentages of Cho
in the corresponding feeding group webs. In some instances, even μg Cho
web–1 was greater in starving groups than in the
corresponding feeding group webs (supplementary material Figs S3–S6 and
Tables S4, S5). This contrasted with what we generally observed: greater
absolute quantities of LMM per web (often considerably so) in feeding groups
than in starving groups due to the usually greater total web mass
(Fig. 1, supplementary material
Figs S3–S6) and mass of water-soluble fractions in feeding groups. In
both Argiope species, negative correlations between web collection
number and Cho + Bet were not significant in starving groups
(Table 5) primarily due to
unexpected end-of-series increases in Cho and Bet. In starving A.
cavaticus groups (juveniles especially), Bet molar percentage decreases
occurred only after initial unanticipated increases
(Fig. 4). Comparable increases
were not seen in feeding A. cavaticus. Also contrary to expectations,
resumed feeding of starving spiders yielded little or no resurgence in Cho and
Bet.

Of the remaining three organic LMM measured in this study, Put and Tau were
invariably minor components (<2 mole%) and free Pro's contribution exceeded
2 mole% only in some A. cavaticus webs
(Table 2). Each went undetected
in all webs from one of the three study species, though it may be that small
quantities were not detected because of overlapping peaks from other
compounds, including minor unidentified compounds. It now appears that the
compound identified as Tau in earlier analyses of A. cavaticus webs
(Townley et al., 1991) was not
Tau but a minor as yet unidentified compound. Shifts in Pro were reminiscent
of Gly with upward trends in both starving and feeding A. cavaticus,
and a drop when feeding was resumed by starving spiders (supplementary
material Figs S3–S5). Absolute quantities of Pro per web were generally
maintained or increased in starving A. cavaticus (supplementary
material Table S4).

Post-egg-sac (PES) webs

The construction of up to three egg sacs by some Argiope aurantia
and Argiope trifasciata and the building of orb webs after each of
these gave us the opportunity to examine changes in LMM composition in webs
built between egg laying episodes by feeding spiders (though, as throughout
this study, determining LMM composition meant that spiders were not allowed to
recycle webs). Cyclical changes in LMM molar percentages were evident when the
data from successive PES groups were examined
(Fig. 5; absolute quantities of
LMM shown in supplementary material Fig. S7). To mention just two examples, in
both species Gly was relatively low following egg sac construction, but
increased subsequently and then declined with the approach of the next
oviposition. Cho, on the other hand, was highest in the first webs built after
an egg sac was made and tended to decline as the series progressed.

When the molar percentages of the positively charged organic LMM (Put, Cho,
GAB and NAP) were added (doubling Put because of its +2 charge) and compared
with the sums of the negative organic LMM (NAT and Ise) there was an `excess'
of positive charge in all 311 web collections analyzed during this study. This
excess was greatest in Argiope aurantia (43.6±0.70 mole%, mean±
s.e.m.; N=102) and least in Argiope trifasciata
(21.5±0.65 mole%; N=123), with Araneus cavaticus
intermediate (31.0±0.86 mole%; N=86). Analyzing the data from
each group separately revealed a highly significant (P≤0.001)
increase over the series in the excess positive charge in all five starving
groups and these increases were greater than and significantly different
(P≤0.05) from increases seen in any other groups. In A.
cavaticus, there was no significant difference between the starving adult
group and either of the starving juvenile groups. Much of the increase in
excess positive charge in the starving groups was due to a drop in negatively
charged organic LMM (NAT+Ise) over the series rather than to an increase in
positively charged organic LMM. In the three starving A. cavaticus
groups, >91% of the increased excess positive charge was due to a decrease
in NAT+Ise. For the A. aurantia and A. trifasciata starving
groups, this percentage was 74% and 48%, respectively.

All web extracts were assayed for inorganic phosphorus to estimate
inorganic phosphate's contribution as a counterion to the excess positive
charge. In only 9 of the 311 web collections did the molar percentage of
H2PO –4 essentially balance or even
slightly exceed the molar percentage of excess positive charge (six of the
nine were in the A. trifasciata resumed feeding series and were due
primarily to the relatively rapid recovery of Ise near the start of the series
decreasing the excess positive charge). In the remaining 302 web collections
the molar percentage of H2PO –4 was
insufficient to balance the molar percentage of excess positive charge. On
average, the percentage of the excess positive charge balanced by inorganic
phosphate was least in the species with the greatest excess positive charge,
A. aurantia (36.3%), and about equal in the other two species (A.
trifasciata 47.7%, A. cavaticus 48.8%). As noted above, the 12
LMM quantitated in this study do not provide a complete inventory of the LMM,
especially of inorganic ions. And a more complete accounting of charges in
this system should also consider components other than LMM, such as the
nodular glycoprotein (see Introduction).

There was a positive correlation between the molar percentages of
H2PO –4 and excess positive charge when
the data from all three species were pooled (r=0.651,
P<0.001, N=311), but only A. cavaticus
(r=0.546, P<0.001, N=86) and A.
trifasciata (r=0.386, P<0.001, N=123)
showed significant correlations when each species was analyzed separately. In
A. cavaticus, this correlation was attributable to a negative
correlation between H2PO –4 and
negatively charged organic LMM (NAT+Ise) (r=–0.643,
P<0.001), as opposed to the situation in A. trifasciata
in which there were significant correlations between H2PO
–4 and both negatively charged
(r=–0.235, P=0.009) and positively charged
(r=0.274, P=0.002) organic LMM. The inverse relationship
between H2PO –4 and the two sulfonic
acids was mostly clearly exhibited in juvenile A. cavaticus (males
and females) and contributed to significant inverse correlations between
H2PO –4 and both NAT and Ise in A.
cavaticus (Table 4).

Discussion

Synthesis of sticky droplet organic LMM

Radiolabeled glucose and methionine/cysteine were fed to spiders to assess
their ability to synthesize sticky droplet LMM. Label from
[14C]glucose appeared in high specific radioactivity in web droplet
Ala, Gly and GAB, while only a meager amount was associated with Cho
(Table 1), indicating that Cho
is nutritionally essential. Ise, Tau (free and as a component of NAT) and the
Put moiety of NAP occupied positions between these extremes, with the
relatively low specific activities of the two sulfonic acids in particular
raising the possibility that, without adequate dietary intake of these LMM (or
more immediate precursors), the spider's synthetic rate may not always be
sufficient to meet the requirements for optimal web construction. These
results are essentially in agreement with the literature, as detailed in the
following sections.

In an earlier study with N. clavipes webs
(Higgins et al., 2001), molar
percentages of free Gly and Ala tended to be positively correlated. Likewise,
in this study, Gly and Ala were positively correlated in the three species
and, in A. cavaticus and Argiope aurantia, Pro was
positively correlated with both Gly and Ala
(Table 4) (Pro was not detected
in webs of A. trifasciata).

Changes in LMM composition in webs built after construction of first (PES
1), second (PES 2), and third (PES 3) egg sacs by Argiope aurantia
(A) and Argiope trifasciata (B). The PES 3 series from A.
aurantia had to be ended prematurely (see Materials and methods). The key
identifies the LMM, arranged in the same order, represented in the bar charts.
Note that some LMM were either not detected in individual web collections or
too minor to be visible in these plots. Glycine betaine and taurine, though
not clearly visible in the bar charts, have been retained in the key as a
reminder that these LMM were detected in some of these web collections and for
consistency with Fig. 1 and
supplementary material Figs S2–S7. GAB, GABamide; Gly, glycine; Ise,
isethionate; NAP, N-acetylputrescine; NAT, N-acetyltaurine;
PES, post-egg-sac group.

NMR analyses have not revealed NAP in webs of N. clavipes, but Put
is present in quantity (Higgins et al.,
2001), as it is in some other araneoids [e.g. Metepeira
incrassata F.O.P.-Cambridge 1903
(Higgins et al., 2001),
Micrathena gracilis (Walckenaer 1805) (M. A. Townley and E. K.
Tillinghast, unpublished)]. Put was radiolabeled in some webs built by N.
clavipes fed [14C]glucose or [14C]acetate, but not
as consistently as GAB, Gly, and Ala
(Higgins and Rankin, 1999).
Likewise, in this study the specific activity of the Put component of NAP was
significantly lower than the specific activities of GAB, Gly and Ala
(Table 1).

In the present study, the specific radioactivity of Bet was not determined
because of suspected contamination, but our observations indicate that
labeling of this compound was slight at most. An earlier study found that Bet
from webs of N. clavipes fed [14C]glucose or
[14C]acetate was not radiolabeled and that this compound is likely
essential (Higgins and Rankin,
1999). A significant positive correlation between molar
percentages of Bet and Cho was reported in one population of N.
clavipes (Higgins et al.,
2001) and was likewise observed in all three species used in this
study (Table 4). Such
observations are consistent with Bet synthesis from Cho via betaine
aldehyde, to our knowledge the only established pathway to Bet in both
chelicerate (Dragolovich and Pierce,
1994) and mandibulate
(Bilinski, 1960;
Weiher and Komnick, 1997)
arthropods.

Is there agreement between synthetic capacity and LMM compositional
changes with starving?

Assuming an organic LMM's specific radioactivity is a reliable indicator of
the spider's ability to synthesize that LMM, we anticipated that, with
fasting, there would be decreased molar percentages of those LMM showing lower
incorporation of radioisotope. Was this expectation met? In some respects,
yes. Downward trends in starving spiders were most evident among `poorly
synthesized' (Cho, Bet) and `moderately synthesized' (Ise, NAT, NAP, Put) LMM,
with the exception of the decline in Ala in A. trifasciata. Extended
upward trends were seen only among `readily synthesized' LMM (GAB, Gly and, to
a far lesser extent, Ala in A. cavaticus) and Pro (presumed at least
a moderately synthesized LMM). Even more than increases in molar percentage,
increases in μg web–1 of Gly, GAB and Pro, as seen over at
least part of a starving series in one or more species, suggest that starving
spiders use these LMM to compensate to some extent for decreases in less
readily synthesized and less available LMM.

Argiope trifasciata provided a particularly striking example of a
compositional shift accompanying starvation that may have resulted from
differences in synthetic capacity; specifically, the decline in molar
percentage of NAP and coincident increase in GAB. In webs of this species, the
mean specific radioactivity of GAB was almost four times higher than that of
the Put moiety of NAP (Table
1). Unlike webs of A. cavaticus and A. aurantia,
webs of A. trifasciata contain NAP as a major constituent whereas GAB
is generally less abundant (Townley et
al., 1991) and sometimes even a minor component, as at the start
of the feeding and starving series (Figs
1,
2). This suggests that these
two similar compounds may fulfill the same, as yet unknown, function in the
sticky droplets and that when A. trifasciata are starved and their
webs removed the more readily synthesized GAB is increasingly recruited to
stand in for NAP. An inverse correlation between molar percentages of NAP and
GAB in A. trifasciata (and A. cavaticus) is consistent with
this interpretation (Table
4).

There were, on the other hand, a number of trends in the webs of starving
spiders that did not conform to expectations based on specific
radioactivities. Ala's unexpected decline in starving A. trifasciata
has already been mentioned, but even the slight molar percentage increases in
Ala in A. cavaticus and A. aurantia were scarcely
commensurate with its specific radioactivity. Perhaps Ala is more in demand in
starving spiders than Gly [e.g. as a substrate for gluconeogenesis
(Felig, 1973)] and, thus, is
less available for use in the web. We also did not anticipate the initial rise
in Bet or NAT at the start of the series in starving A. cavaticus,
when mass of the water-soluble fraction was already on the decline, nor the
resurgence in Bet at the end of the series in both Argiope species.
Also unexpected, Ise partially rebounded at the end of the starving A.
trifasciata series, but the other major sulfonate, NAT, did not.
Conversely, NAT partially rebounded at the end of the starving A.
aurantia series, but Ise did not (Fig.
3). Neither rebounded in A. cavaticus. Probably not
coincidentally, Ise is generally more abundant than NAT in webs of A.
trifasciata while the opposite is true of A. aurantia webs
(Vollrath et al., 1990;
Townley et al., 1991). Given
Cho's very low specific radioactivity, we were especially surprised that,
after an initial decline, its molar percentage tended to level out higher than
in the corresponding webs of the feeding groups. If the spiders' ability to
synthesize Cho is so poor, where was it coming from in the starving spiders?
One possibility is the store of Cho residing in membrane phospholipids,
mobilized as tissue reserves were broken down to meet the energy and material
needs of vital tissues.

The most unexpected results, however, came from webs of the feeding
controls. LMM composition was not as stable in these webs as we had
anticipated. As detailed above, some compositional shifts in feeding group
webs, especially of Gly, GAB, Pro, Cho, Bet, Ise and Put, were similar to
trends observed in starving groups. Thus, some changes in composition with
starvation are likely attributable, at least in part, to factors other than
starvation shared by feeding group spiders. One such factor that almost
certainly contributed to similar trends was the absence of web recycling,
discussed in the following section. Another possibility, however, is that
starving and feeding spiders were both responding to limited resources, but
arising for different reasons; an absence of prey in starving groups
versus allocation of resources to other activities, or the allocation
of more resources to web building, in feeding groups. Examples of other
activities include molting in juveniles and egg laying in adult females. These
different possibilities are not mutually exclusive and the relative importance
of each may differ among the different LMM.

For the present we are proceeding from the assumption that shifts in LMM
composition seen in starving groups were shifts away from what would generally
be a more effective composition for securing prey. This assumption may not be
correct. At present we know almost nothing about how LMM compositional
differences affect sticky spiral functioning.

Possible effects of web recycling on LMM composition

Spiders in the field often have the opportunity to recycle at least a
portion of their old orb web by ingestion before they construct a new one
(Hingston, 1922;
Peakall, 1971;
Carico, 1986 and references
therein; Craig, 1989). In this
study, however, spiders were only allowed to recycle their first post-ecdysial
web; all other webs were collected for analysis. Web recycling or its lack
clearly influences some web parameters
(Breed et al., 1964) and this
influence appears to extend to LMM composition. It seems likely that similar
trends seen between feeding and starving groups were at least partly the
result of these spiders being deprived of web material, particularly LMM, that
they normally would have been able to recoup. It is especially likely that
some differences seen between earlier and later web collections within a
series were related to web recycling having occurred just before the start of
the series, but not subsequently.

For example, we have evidence that the relatively steep decline in Cho
early in the series in feeding and starving groups was the result of webs
being recycled before, but not after, the start of the series. This evidence
comes from an experiment in which we fed 4 male and 4 female penultimate
instar A. cavaticus a solution containing 6.54×106
c.p.m. [1,2-14C]Cho chloride (NEN, Boston, MA, USA) and 43.9 μg
Cho. These spiders had all built two post-ecdysial webs, both of which we had
removed, and had not been fed since prior to ecdysis. After radioisotope
feeding, spiders received one fly (P. sericata or P. regina)
after each of the first four webs built. We found that 76.6±2.42% (mean±
s.e.m., N=8) of the 14C ingested was present in
the water-soluble fraction of the first 2 webs built; 81.5±2.59% was
present in the first five webs built. No significant difference was found
between males and females comparing data from the first two webs
(P=0.646) or webs 3–5 (P=0.265; unpaired
t-test). Using the same protocol, we also attempted to feed four
males and one female the same amount of [14C]Cho to which an
additional 224 μg unlabeled Cho was added, but only one male drank the
solution quickly and without incident. The other spiders did eventually drink
comparable, but imprecisely known, volumes. Nevertheless, with the one
cooperative male, 75.0% of the ingested 14C was present in the
first two webs built; 78.3% was in the first six webs built. The results from
the other four spiders, while only approximate, indicate that this result is
representative.

Thus, it appears that a large percentage of ingested free Cho, such as the
spider receives when it recycles an old web, is incorporated into future webs,
with the bulk of this going into the first web. This high percentage of
incorporation can occur even when relatively large quantities of free Cho (268μ
g) are consumed, resulting in webs with unusually high molar percentages
of Cho. For example, we analyzed the first labeled web built by the one female
fed [14C]Cho spiked with unlabeled Cho. Based on the 11 organic LMM
constituting 100% of the LMM, Cho accounted for 60.6 mole% of the LMM in this
web (!), much higher than we have ever seen in webs built by spiders fed only
insects. By the second web, which contained only 13.8% as much 14C
as the first web, Cho had dropped to a more typical 11.8 mole%.

Considering the large contribution made by LMM to total web mass (see
`Gravimetric data' in Results) and the spider's limited capacity for
synthesizing some of the LMM, the principal selective advantage in web
recycling behavior may come from retrieval of LMM rather than silk protein
residues (T. A. Blackledge, personal communication). As further suggested to
us by Blackledge, this possibility is supported by observations indicating
that those orb weavers that build webs lacking sticky droplets tend not to
recycle their webs (e.g. the araneids Cyrtophora and
Mecynogea (Lubin,
1986; Carico,
1986), and some, though apparently not all, uloborids
(Eberhard, 1971;
Opell, 1982;
Lubin, 1986;
Watanabe, 2001). However, the
benefits from consuming water, very small insects
(Nentwig, 1985) or pollen
(Smith and Mommsen, 1984) when
recycling webs cannot be discounted.

Possible effects of the molt/intermolt and egg laying cycles on LMM
composition and web mass

We anticipated that the feeding group spiders might be less than ideal
controls since they would be more apt to molt and lay eggs than starving group
spiders. Indeed, most starving group juveniles did not molt and no starving
group adults oviposited until feeding was resumed whereas all feeding group
juveniles molted and some feeding group adults, though unmated, oviposited
(supplementary material Fig. S1). If these factors affect the allocation of
LMM to the web, then differences seen between feeding and starving groups may
not be attributable solely to changes resulting from starvation. For example,
as noted earlier, the molar percentage of Cho tended to be higher in starving
groups than in feeding groups as series progressed and in some of these later
web collections even μg Cho web–1 was higher in the
starving groups. Was this difference due entirely to starvation, with free Cho
liberated from membrane phospholipids as starving spiders tapped tissue
reserves, or was it to some extent due to Cho in feeding spiders being
diverted into reproduction or growth/molting?

Support for the second of these possibilities was provided by webs of PES
group Argiope and juvenile Araneus cavaticus. In both
Argiope species, Cho levels were highest in the first or second webs
built after an egg sac was constructed. They dropped again with the approach
of the next egg sac's construction (Fig.
5), suggesting that available Cho was being diverted away from
foraging and into reproduction. In juvenile male and female A.
cavaticus, μg Cho web–1 were lower in the last web
collections from feeding spiders than in the corresponding webs from starving
spiders (supplementary material Table S4), suggesting that Cho was being
diverted or held in reserve due to the impending molt.

The influence of reproduction and molting on LMM composition apparently
extends beyond the above example with Cho. Cyclical changes in quantities of
other LMM, synchronized to the egg laying cycle, are also evident in the data
from the PES groups (Fig. 5).
Certain end-of-series departures from earlier trends seen in feeding adults,
but not feeding juveniles, such as declines in Gly, Ala, and Pro
(Fig. 2), may also reflect the
influence of egg laying. And in the latter half of the series from the A.
cavaticus feeding juvenile male group (supplementary material Fig. S3),
the drop in μg web–1 of several LMM indicates that the
reallocation of resources away from foraging applies to LMM in addition to
Cho.

Growth/molting and reproduction may have contributed not only to
differences between the webs of feeding and starving groups, but also to
similarities. The reallocation of LMM due to growth/molting or reproduction in
feeding groups may have produced shortages for web construction that in some
ways resembled the effects of starvation. Perhaps some of the same changes in
LMM composition made necessary by starvation are also favored in some
circumstances by feeding spiders endeavoring to lay eggs or ecdyse.
Reallocation of resources may be most evident in spiders subsisting on a diet
that is quantitatively or qualitatively suboptimal, a topic we consider
below.

The molt/intermolt and egg laying cycles also bring about changes in other
web parameters, including, as seen in previous studies, web size, and as seen
in this study, web mass. In a study with juvenile Nephila clavipes
(where webs were not removed), orb web size typically increased in the earlier
part of an intermolt and then decreased with the approach of the next ecdysis
(Higgins, 1990). It has
likewise been noted that A. aurantia build smaller webs around the
time of molting (Reed et al.,
1969). In our feeding juvenile A. cavaticus (where webs
were removed), web mass did not change substantially in the earlier part of
the intermolt, but a clear decline in web mass with the approach of ecdysis
was seen in males, though not females (supplementary material Figs S3, S4).
Presumably, this difference between the sexes reflects a major difference
following the final molt; males are unable to build orb webs
(Sekiguchi, 1955) and thus
much less likely to feed as adults than females. An initial increase in web
mass in feeding adults, especially Argiope
(Fig. 1, supplementary material
Fig. S6), following the final molt may also reflect the influence of the
molt/intermolt cycle. In N. clavipes
(Higgins, 1990) and
Larinioides cornutus (Clerck 1757)
(Sherman, 1994), web size
declined with the approach of egg laying. In this study, measurements of web
mass indicated the same trend, seen most convincingly in the two
Argiope species (the two species that produced egg sacs in the
laboratory). Following oviposition, web size has been observed to increase in
L. cornutus (Sherman,
1994) and we likewise observed web mass rebound in the two
Argiope species after an egg sac was made (supplementary material
Fig. S7).

The changes in web parameters apparently associated with the molt/intermolt
and egg laying cycles may be considered in terms of resource allocation, with
relative investments in foraging (the web primarily), growth and reproduction
changing over time. But with molting and ovipositing, the influence of more
tangible, anatomical changes should be considered as well. Several types of
silk glands are remodeled during a molt, including those that produce the
sticky spiral, the aggregate and flagelliform glands. At the height of
remodeling these silk glands are nonfunctional
(Townley et al., 1993) and orb
web construction ceases for up to several days before ecdysis (e.g.
Witt, 1971;
Higgins, 1990). But beyond
this clearcut effect on web building, it is possible that the last webs built
before ecdysis and/or the first webs built after are the products of silk
glands that are in the earliest or final phase of remodeling, respectively. If
so, changes taking place in these silk glands might put constraints on certain
web parameters such that some structural or compositional options, available
at other times, are not available close to ecdysis.

There are also anatomical changes in the silk glands associated with egg
development. It is not unusual for a large part of the abdomen to become
increasingly crowded by eggs and cylindrical silk glands (major sources of egg
sac silk) during this time. As a result, other tissues become compressed,
including the aggregate glands, source of the web's sticky droplets. This
compression may render these and other silk glands temporarily nonfunctional,
forcing a suspension of web building until oviposition
(Kovoor, 1972;
Kovoor, 1977). Thus,
comparable to silk gland remodeling during molting, the structure and
composition of last webs built before oviposition may reflect silk glands
experiencing reduced functionality.

Note that both molting and egg laying are often accompanied by a hiatus in
web building extending before and/or after the day on which ecdysis or egg sac
construction occur. This effect on the time between bouts of web building may
itself influence LMM composition in the first post-ecdysial or
post-ovipositional webs by affecting quantities of LMM that can be synthesized
or otherwise amassed for use in these webs.

Possible effects of feeding regimen on LMM composition among feeding
group spiders

When setting the feeding regimen for the feeding groups it was our
intention that spiders should not gain mass so fast that juveniles molted or
adults oviposited after building only a few webs. There was a tendency for web
mass to increase as the series progressed in feeding group spiders,
particularly in the two Argiope species. How did the feeding regimen
affect decisions regarding foraging investment? Did maintained or increased
web mass resulting from such decisions contribute to compositional shifts
similar in some respects to those seen in starving groups? We consider and
provide some background to these questions in the following paragraphs.

Thus, was the tendency for web mass to increase in feeding group spiders,
especially Argiope, an indication that the feeding rate was only
moderate, a condition exacerbated by our removal of webs, and consequently
foraging investment was increased? Or was the feeding rate more than moderate,
resulting in a response (increased web mass) that would allow the spiders to
better exploit the relatively abundant prey
(Benforado and Kistler, 1973;
Vollrath and Samu, 1997)? Or
was increased web mass a response to a consistent, as opposed to a sporadic,
return on the foraging investment
(Herberstein et al., 2000)?
There is also the possible influence of the molt/intermolt cycle on web mass,
mentioned earlier. Whichever, if any, of these four explanations apply, such
increases in web mass may have spread certain resources too thin, such that
compositional compromises were made by feeding group spiders. If resources
were not only lost in the webs we collected, but were increasingly diverted
into reproduction and/or growth by feeding group spiders, it would presumably
have been even more difficult to maintain an optimal LMM composition while
maintaining or increasing web mass. Thus, certain shifts in composition in
feeding groups (e.g. the growing molar percentage of Gly) that were mirrored
in starving groups may have reflected a compromise made by feeding spiders
that enabled them to build heavier webs. Whether because of dietary
deficiencies, synthetic capacity limitations, or diversion of resources into
reproduction or growth, it may not always be possible to allocate the `ideal'
quantities of LMM to webs containing more aggregate gland secretion. Perhaps
it is sometimes more advantageous to build heavier webs with a less-than-ideal
LMM composition than to build lighter webs with the `ideal' LMM
composition.

Indeed, compositional compromises made under certain circumstances have
previously been indicated in non-sticky web components. One study found that
starvation may result in decreased extensibility of major ampullate silk
(Madsen et al., 1999). They
suggested that decreased availability of amino acids with starvation may
result in compromised silks with compositions and mechanical properties
different from those of well-nourished spiders. A related suggestion was made
based on data indicating that qualitative differences in diet can influence
the amino acid composition of major ampullate silk
(Craig et al., 2000). The
observation of considerable intraspecific and intraindividual variability in
major ampullate silk amino acid composition lends support to these
possibilities (Work and Young,
1987 and references therein;
Craig et al., 2000), while the
observation of a uniform intraspecific composition does not
(Lombardi and Kaplan,
1990).

In comparing our study to earlier studies we should bear in mind
differences in the web parameters measured. In this study, web mass was
measured but dimensional parameters such as web size and mesh size were not,
while in most earlier studies the converse is true. Ideally, the monitoring of
foraging investment would include measures of both to help prevent or resolve
seeming contradictions that can arise between studies. For example, in this
study decreases in web mass were observed soon after starvation and web
removal began, whereas in an earlier study no significant decrease in orb web
size was observed after 6 days of starvation and web removal
(Witt, 1963). And with
resumed feeding of starved spiders there was no significant increase in web
size even after 10 days of feeding (Witt,
1963), again in contrast to our mass results. However, Witt also
obtained a measure of web mass in the form of total web nitrogen content
which, divided by total thread length, was used to calculate `thread
thickness'. Witt notes that, despite web size remaining large after 6 days of
starvation, there was a decrease in thread thickness in these webs, consistent
with our mass data. Parenthetically, given the large contribution the sticky
spiral's aggregate gland cover makes to orb web mass and the recognition that
many of the organic LMM contain nitrogen, we suggest that changes reported in
`thread thickness' with different feeding and/or web removal protocols
(Witt, 1963;
Breed et al., 1964) may be
partly or largely attributable to changes in the quantity of aggregate gland
secretion applied per length of sticky spiral. In our study, the tendency for
the water-soluble percentage of the web to decrease over the series in many
groups (supplementary material Table S3) indicates a decrease in this
quantity. More direct observations have confirmed that this quantity varies,
even within a single web (Eberhard,
1988; Vollrath and Edmonds,
1989; Edmonds and Vollrath,
1992).

In discussing the influence of the feeding regimen on compositional changes
we should consider not only feeding rate but also quality of the diet. Of the
three species used in this study, A. cavaticus is probably the most
reliant on dipterans as a natural source of food (e.g.
Olive, 1980;
Riechert and Cady, 1983;
Horton and Wise, 1983;
Howell and Ellender, 1984).
But even in this species a diet consisting of just one or a few species of
brachyceran flies, as given to all feeding group spiders, is not typical of
spiders in the field. Perhaps the same quantity of food, but from more varied
or typical prey items, would have resulted in more stable LMM compositions in
feeding group spiders. Qualitative aspects of the diet may also have
contributed to differences in composition seen between radiolabeled and
non-radiolabeled webs. As noted earlier, the molar percentage of Cho in the
webs of Argiope given radioisotope was invariably higher than in the
non-radioactive webs of conspecifics. The former had either recently been
feeding in the field or were fed orthopterans, whereas the latter were fed
only muscid and calliphorid flies. We note that a significant difference in
the amino acid composition of A. keyserlingi dragline silk has been
observed depending on whether the spiders had been feeding on blowflies or
crickets (Craig et al.,
2000).

Consistency amidst variation

While recognizing the significant intraspecific variation that clearly
exists in LMM composition, and the validity of the statement made earlier
(Higgins et al., 2001), that
`the composition of the organic low-molecular-weight solution is not
fixed', we should also recognize that there are features to a species'
LMM composition that are at least typically maintained. The inventory of
principal LMM is generally consistent within a species, at least within one
sex, and some quantitative relationships among the LMM are often observed. In
this study, GAB was invariably the most abundant LMM in webs of A.
aurantia and A. cavaticus on a molar percentage basis
(supplementary material Fig. S8). In A. cavaticus webs the molar
percentage of NAT was almost invariably lower than that of Ise, often by a
considerable margin (supplementary material Figs S3–S5), while the
opposite was observed in A. aurantia
(Fig. 5, supplementary material
Fig. S6). Some quantitative relationships were observed in all three species.
For example, Cho was usually (A. trifasciata) or always (A.
aurantia, A. cavaticus) more abundant than Bet, and Gly was usually
(A. cavaticus) or always (A. trifasciata, A. aurantia) more
abundant than Ala or Pro. Many other such relationships were demonstrated by
ranking molar percentage data for each species (supplementary material Fig.
S8).

Conclusions

We investigated how starvation affects LMM composition in orb web sticky
droplets, anticipating that with fasting there would be decreases in the molar
percentages of those organic LMM the spider is least able to synthesize and
increases in those that are more readily synthesized. Many shifts in
composition were basically consistent with differing synthetic capacities.
Thus, declines in the molar percentages of LMM with lower rates of synthesis
(Cho, Ise, NAT and, presumably, Bet) were observed at least over part of a web
series in starving group spiders, while molar percentages of some LMM with
higher rates of synthesis increased (Gly, GAB and, in Araneus
cavaticus, Pro). The most convincing indications that certain more
readily synthesized LMM were increasingly relied upon by starving spiders were
increases observed in μg web–1 of Gly, GAB (in Argiope
trifasciata) and Pro (in A. cavaticus) in webs of starving group
spiders. These increases in absolute quantities per web contrasted sharply
with the decreases seen in most LMM in starving spiders and give the
impression that these LMM were used to fill in for other, perhaps more
desirable, but unavailable or costly LMM. In A. trifasciata, it
appeared that GAB was increasingly used to substitute for the less readily
synthesized NAP.

However, synthetic capacity was not an entirely reliable predictor of
compositional changes with starvation. Some shifts in LMM molar percentages in
starving group spiders were not predicted based on specific radioactivity
measurements (e.g. the decrease in Ala in A. trifasciata). We also
found a number of similarities between starving and feeding group spiders with
respect to changes in LMM molar percentages, making interpretation of the
results uncertain. Possible explanations for these parallel changes include a
factor common to both the feeding and starving groups. It is likely that one
such factor was web recycling. By not allowing spiders to recycle their webs,
a form of nutritional stress was imposed on feeding as well as starving group
spiders and may have influenced LMM composition in a similar manner in both.
It may also have contributed to decreases observed both in the water-soluble
percentage of the web and the percentage of water-soluble mass that could be
accounted for by the organic LMM. Paradoxically, it is also possible that the
difference imposed on feeding and starving group spiders, availability of
insect prey or lack thereof, contributed to similar molar percentage shifts.
Feeding group spiders allocated resources to molting, egg laying and increased
foraging (increased web mass), and these expenditures may have resulted in
shortages of some LMM and consequent shifts in LMM composition, reminiscent of
shifts due to fasting in starving group spiders. Analyses of webs of juveniles
and adults indicated that both the molt/intermolt and egg laying cycles,
respectively, influenced LMM composition.

Additional studies are needed to focus on individual factors that probably
influence LMM composition, including those touched on in this report (web
recycling, the molt/intermolt and egg laying cycles, qualitative and
quantitative aspects of diet, time between web building episodes), as well as
studies that focus on the synthesis, allocation, and transport of individual
LMM.

List of abbreviations

Ala

alanine

Bet

glycine betaine

Cho

choline

2D-TLC

two-dimensional thin layer chromatography

GAB

4-aminobutyramide

Gly

glycine

1H-NMR

proton nuclear magnetic resonance spectroscopy

HVE

high-voltage paper electrophoresis

Ise

isethionic acid (2-hydroxyethane sulfonic acid)

LMM

sticky droplet low-molecular-mass compounds

NAP

N-monoacetylputrescine

NAT

N-acetyltaurine

PES

post-egg-sac

Pro

proline

Put

putrescine

Tau

taurine

ACKNOWLEDGEMENTS

This study would not have taken place without the aid of Kathy Gallagher
(UNH Instrumentation Center), who went well above the call of duty in placing
her expertise with NMR at our disposal, providing training and making large
blocks of instrument time available to us. Nor would this study have been
possible without partial financial support from the National Institutes of
Health, grant R15DK/OD51291, to E.K.T. We are also very grateful to Dr Stacia
Sower (UNH) for the use of laboratory equipment and to Dr Todd Blackledge
(University of Akron) and an anonymous reviewer for their many valuable
suggestions for improving the manuscript.

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